Anti-tumor fibrillar human serum albumin methods and compositions

ABSTRACT

Fibrillar human serum albumin was shown to be effective in the treatment of various types of cancers. Methods and compositions are disclosed for using fibrillar human serum albumin as a medicament to treat subjects having cancer.

This application is a divisional application of U.S. patent applicationSer. No. 12/623,162 that has been patented as U.S. Pat. No. 8,357,652.

BACKGROUND

The present disclosure is related to making and using fibrillar humanserum albumin. (HSA) Fibrillar HSA was shown to cause apoptosis of manytypes of cancer cells by modulating the Akt signaling pathway, asdisclosed in U.S. Pat. No. 7,488,800, which is incorporated byreference.

SUMMARY

Fibrillar human serum albumin was shown to be effective in the treatmentof various types of cancers. Methods and compositions are disclosed forusing fibrillar human serum albumin as a medicament to treat subjectshaving cancer.

According to a feature of the present disclosure, a composition isdisclosed comprising fibrillar human serum albumin and apharmaceutically acceptable carrier.

According to a feature of the present disclosure, a method is disclosedcomprising administering to a subject having cancer a therapeuticallyeffective amount of fibrillar human serum albumin.

According to a feature of the present disclosure, a method is disclosedcomprising manufacturing a composition useful in the treatment ofcancer, the medication comprising fibrillar human serum albumin and apharmaceutically acceptable carrier.

According to a feature of the present disclosure, a method is disclosedcomprising providing a therapeutically effective amount of fibrillarserum albumin for use in a subject having cancer.

According to a feature of the present disclosure, a method is disclosedcomprising dissolving HSA in an SDS solution; applying the dissolved HSAthrough a gel filtration column with a pore size of at least about 70kDa; removing the HSA from the column; and dialyzing the solutionagainst phosphate buffered saline to remove the SDS.

DRAWINGS

The above-mentioned features and objects of the present disclosure willbecome more apparent with reference to the following description takenin conjunction with the accompanying drawings wherein like referencenumerals denote like elements and in which:

FIG. 1 is an implementation of experimental data showing a comparison offluorescence level of increasing concentrations of F-HSA, HSA, and A β(1-42) after incubating with 20 μM amyloid-specific dye ThT for 1 h;

FIG. 2 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on breast cancer cells;

FIG. 3 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on breast cancer cells;

FIG. 4 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on ovarian cells;

FIG. 5 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on cervical cancer cells;

FIG. 6 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on prostate cancer cells;

FIG. 7 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on prostate cancer cells;

FIG. 8 is an implementation of experimental data showing the cytotocixeffects of F-HSA on lung cancer cells;

FIGS. 9A-9H are implementations of experimental data showing the effectof F-HSA in reducing the rate of tumor cell migrations and invasionwithout effecting viability of normal cells;

FIGS. 10A-10C are implementations of experimental data showing theeffect of F-HSA in suppressing the metastasis of mouse breast tumor TS/Acells to the lung; and

FIGS. 11A-11C are implementations of experimental data showing theeffect of F-HSA in suppressing the metastasis of mouse breast tumorMDA-MB-231 cells to the lung.

DETAILED DESCRIPTION

In the following detailed description of embodiments of the presentdisclosure, reference is made to the accompanying drawings in which likereferences indicate similar elements, and in which is shown by way ofillustration specific embodiments in which the present disclosure may bepracticed. These embodiments are described in sufficient detail toenable those skilled in the art to practice the present disclosure, andit is to be understood that other embodiments may be utilized and thatlogical, mechanical, electrical, functional, and other changes may bemade without departing from the scope of the present disclosure. Thefollowing detailed description is, therefore, not to be taken in alimiting sense, and the scope of the present disclosure is defined onlyby the appended claims. As used in the present disclosure, the term “or”shall be understood to be defined as a logical disjunction and shall notindicate an exclusive disjunction unless expressly indicated as such ornotated as “xor.”

This application incorporates by reference U.S. Patent ApplicationPublication No. 2008/0300186, published Dec. 4, 2009.

The present disclosure relates to a process of producing fibrillarproteins and methods of treatment using fibrillar proteins. This processhas advantages which include ease of control, homogeneity of production,and feasibility of scaling up. Moreover, fibrillization of proteins canbe induced by this process without the assistance of fibril seed. Even atiny amount of protein would be applicable to this process. As usedherein, “protein” includes one or more proteins, protein fragments,polypeptides, or peptides. Proteins include both synthetic and naturallyoccurring proteins.

According to the present disclosure, a method is disclosed for changinga globular protein structure into a fibrillar protein structure. Themethod can be used to convert native proteins, regardless of theirsequence, into fibrillar form in a simple and rapid manner. The methodcomprises the steps of dissolving a globular protein in a solution thatcontains detergents and applying the solution to a molecular sizingcolumn that can separate proteins of 70 kDa molecular weight or larger,and eluting the protein with a solution containing detergent.

In an exemplary implementation, the method comprises the steps ofproviding a globular protein, forming a solution containing the globularprotein, adding a detergent to the solution containing the globularprotein, and applying the solution to a molecular sizing column with apore size of at least 70 kDa.

In an exemplary implementation, the method comprises the steps ofproviding a globular protein, forming a solution containing the globularprotein, adding a detergent to the solution containing the globularprotein, and applying the solution to a molecular sizing column with apore size of at least about 70 kDa in the presence of low concentrationof detergent.

Globular proteins, also known as spheroproteins, are one of two maintertiary structure classes of proteins. Globular proteins are generallysoluble and form spheriodal molecules in water. They have a complexsecondary structure comprising a mixture of secondary structure motifs,such as α-helices, β-sheets, and loop structures. The other maintertiary structure class of proteins are fibrillar proteins, or fibrousproteins. Fibrillar proteins are generally insoluble and have anelongated shape. They have a simpler secondary structure and are oftenbased on only one type of secondary structure motif.

Surfactants, also referred to herein as detergents, are substances thatlower the surface tension of water and increase the solubility oforganic compounds. Detergents may be ionic, which includes cationic,anionic, and zwitterionic detergents, as well as non-ionic. Detergentsplay a role in disrupting non-covalent bonds in proteins, therebydenaturing the proteins such that they lose their native shape orconformation. In exemplary implementations, the detergent used is sodiumdodecyl sulfate (SDS), obtained from Sigma. In other exemplaryimplementations, the detergent used is Zwittergent 3-14, obtained fromCalbiochem.

Amyloids are fibrous cross-β protein aggregates. Numerous proteins arecapable of converting to amyloid-like fibrils with characteristics thatinclude fibrillar morphology, protofilament substructure, cross-βdiffraction pattern, an increase in β-structure, Congo red binding, andThT binding. In exemplary implementations, the globular protein isconverted to form amyloid-like fibrils, which allows for the convertedprotein to be identified by its amyloid-like properties.

According to implementations, chromatography may be used in the processto convert the globular protein structure into a fibrillar proteinstructure and separate them. Generally, chromatography is accomplishedusing columns, though other methods such as those used for thin-layerchromatography may also be possible. Chromatography techniques includesize exclusion, affinity, and ion-exchange. Though a batch-typeproduction of fibrillar proteins is possible, utilizing a column processallows globular proteins to be converted into a fibrillar form in arapid, steady, efficient, and continuous manner. Scaling-up this processis also possible with the usage of columns.

According to exemplary implementations, size exclusion chromatographywith bead pore sizes of at least about 70 kDa is used. The bead poresize used may vary depending on various characteristics of the globularprotein, for example its size. The pore size plays a role in allowingproteins to enter the bead matrix, thus leading to mechanical forceswhich contribute to protein unfolding/folding and enhance fibrillogenicensemble. In exemplary implementations, the molecular sizing column usedis a Superdex 200. In other exemplary implementations, the molecularsizing column used is a HW55S.

For column chromatography, a buffer solution containing lowconcentration(s) of detergent may be used to elute the column. Inexemplary implementations, the molecular sizing column is eluted with abuffer solution containing 25 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 MNaCl, and 0.05% SDS. In other exemplary implementations, the molecularsizing column is eluted with a buffer solution containing 25 mMTris-HCl, pH 8.0, 1 mM EDTA, 0.1 M NaCl, and 0.05% Zwittergent 3-14. Theeluant may be collected as fractions and the fractions containing thefibrillar protein subsequently pooled together. The pooled fraction maythen be further filtered to purify and isolate the fibrillar protein,for example dialyzing against PBS to remove SDS or Zwittergent 3-14.

According to implementations, human serum albumin (HSA) can be made intofibrillar human serum albumin by the processes disclosed herein forcreating fibrillar proteins. According to implementations, human serumalbumin has been confirmed convert to fibrillar form by the processesdisclosed herein. With respect to creating fibrillar proteins, U.S. Pat.No. 7,488,800 is incorporated by reference.

The fibrillar HSA (F-HSA) was unexpectedly found to be at least aspotent as recombinant capsid protein of foot and mouth disease virus(rVP1) in causing apoptosis in a variety of cancer cells. Among theadvantages of using F-HSA instead of rVP1 as a cancer therapeutic isthat HSA is a human endogenous protein. Thus, HSA or its derivativeswith similar sequence and composition would be less likely than foreignproteins such as rVP1 to induce immunogenicity and neutralizingantibodies during clinical applications.

According to implementations, F-HSA was generated by dissolving HSA in a1% SDS solution, passing through a Superdex-200 gel filtration columnand eluting with a buffer solution containing 25 mM Tris-HCl (pH 8.0), 1mM EDTA, 0.1 M NaCl, and 0.05% SDS (FIG. 1). After dialysis against PBSto remove the SDS, it was found that unlike HSA, the eluted F-HSA fromthe Superdex-200 column exhibited enhanced fluorescence level ofamyloid-specific dye ThT in a dose-dependent manner (FIG. 1).

It was then determined that F-HSA induced cytotoxicity in cancer cells.As fibrillar serum albumin bound to receptors such as integrins on thecell surface while globular serum albumin could not, it is believed thatthe change of structure of serum albumin from globular to fibrillar formhas enabled the proteins to selectively target cancer cells thatexpressed more integrin α5β1 than normal cells. F-HSA inhibited breastcancer cell growth dose dependently including TS/A (murine mammaryadenocarcinoma) and MDA-MB-231 (human mammary adenocarcinoma) cells withIC₅₀ of 0.15 (FIG. 2) and 0.48 μM (FIG. 3), respectively. F-HSAinhibited ovarian cancer cell SKOV3 growth with IC₅₀ of 0.6 μM (FIG. 4)and cervical cancer cell CaSki growth with IC₅₀ of 1.1 μM (FIG. 5).F-HSA also induced cytotoxicity in prostate cancer cells PC-3 and 22Rv1with IC₅₀ of 0.35 (FIG. 6) and 0.2 μM (FIG. 7), respectively. Inaddition, F-HSA induces cytotoxicity in a number lung cancer cell lines(FIG. 8).

According to implementations, therefore, a method for treating cancer isdisclosed. The method comprises the steps of providing HSA, changing theHSA into a fibrillar structure, and administering a therapeuticallyeffective amount of the F-HSA to a patient in need thereof. Conversionof the HSA into fibrillar form increases its cytotoxic effects on targetcells.

In exemplary implementations, the cancer is a kidney, breast, lung,prostate, liver, cervical, or ovarian cancer. In exemplaryimplementations, the fibrillar HSA plays a role in inducing cancer cellapoptosis by modulating the Akt signaling pathway. In some instances,the fibrillar HSA modulates integrin α5β1 or αvβ3 which leads to thedeactivation of Akt. In other instances, fibrillar HSA binds to integrinand causes cellular apoptosis mainly through theintegrin/FAK/Akt/GSK-3β/caspase-3 pathway.

The fibrillar HSA protein, derivate, ortholog, or other protein havingsubstantial identity to HSA for treating the cancer may be selectedbased on the severity of the disease and the desired cytotoxicity to thecancer cells. In exemplary implementations, for greater cytotoxicity tothe cancer cells, a protein with an RGD motif or greater molecularweight is selected. RGD motif is a ligand for integrins. It has beenshown that fibrillar proteins induced cell death via modulatingintegrin/Akt signaling pathway. It has been found that fibrillarproteins with RGD motifs, like rVP1-S200 and FN-S200, were morecytotoxic than those without RGD motifs such as BSA-S200 and rVP3-S200.

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions, and truncations in the polypeptide encoded by thereference sequence, as discussed herein.

A typical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, and deletions in any combination. Asubstituted or inserted amino acid residue may or may not be one encodedby the genetic code. A variant of a polynucleotide or polypeptide may bea naturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques or by direct synthesis.

An “ortholog” denotes a polypeptide or polynucleotide obtained fromanother species that is the functional counterpart of a polypeptide orpolynucleotide from a different species. Sequence differences amongorthologs are the result of speciation.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity. Preferably, residuepositions which are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleuci-ne, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofcompositions having cytotoxic activities are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid of HSA sequence maintain at least 75%, at least 80%, atleast 90%, at least 95%, or at least 99% identity. In particular,conservative amino acid replacements are contemplated. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare generally divided into families: (1) acidic=aspartate, glutamate;(2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. More preferred families are: serine and threonineare aliphatic-hydroxy family; asparagine and glutamine are anamide-containing family; alanine, valine, leucine and isoleucine are analiphatic family; and phenylalanine, tryptophan, and tyrosine are anaromatic family. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding or properties of the resultingmolecule, especially if the replacement does not involve an amino acidwithin a framework site. Whether an amino acid change results in afunctional peptide can readily be determined by assaying the specificactivity of the polypeptide derivative. Fragments or analogs of proteinsor peptides of the present invention can be readily prepared by those ofordinary skill in the art. Preferred amino- and carboxy-termini offragments or analogs occur near boundaries of functional domains.Structural and functional domains can be identified by comparison of thenucleotide or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Effective amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (5) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmutations of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991), which are each incorporatedherein by reference.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal or carboxy-terminal deletion, but where theremaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long, morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long.

Pharmaceutical or Nutraceutical Compositions

According to another aspect of this disclosure, fibrillar HSA can beincluded in a pharmaceutical or nutraceutical composition together withadditional active agents, carriers, vehicles, excipients, or auxiliaryagents identifiable by a person skilled in the art upon reading of thepresent disclosure.

The pharmaceutical or nutraceutical compositions preferably comprise atleast one pharmaceutically acceptable carrier. In such pharmaceuticalcompositions, the fibrillar HSA or fibrillar HSA equivalent forms the“active compound,” also referred to as the “active agent.” As usedherein the language “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into the compositions. Apharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal,intraperitoneal, intraarterial, intramuscular, intralesional, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycol,or other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates, or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

“Subject” as used herein refers to humans and non-human primates (e.g.guerilla, macaque, marmoset), livestock animals (e.g. sheep, cow, horse,donkey, pig), companion animals (e.g. dog, cat), laboratory test animals(e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animals(e.g. fox, deer), and any other organisms who can benefit from theagents of the present disclosure. There is no limitation on the type ofanimal that could benefit from the presently described agents. A subjectregardless of whether it is a human or non-human organism may bereferred to as a patient, individual, animal, host, or recipient.

Pharmaceutical compositions suitable for an injectable (or an infusion)include sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,Cremophor® EL (BASF, Parsippany, N.J.), or phosphate buffered saline(PBS). In all cases, the composition must be sterile and should be fluidto the extent that easy syringability exists. It should be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.According to embodiments, isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, or sodium chloride in thecomposition are added. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preparation is prepared by vacuumdrying or freeze-drying, which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, or adjuvant materials can beincluded as part of the composition. The tablets, pills, capsules,troches and the like can contain any of the following ingredients, orcompounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a disintegrating agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate or Sterotes; a glidantsuch as colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or strawberry, cherry, grape, lemon, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

According to embodiments, intravitreal injection is accomplished usingPLGA-based microparticles or nanoparticles (liposomes). PEG-basedformulas may also be used. Accordingly, the other methods for injectablepharmaceutical compositions are expressly contemplated for intravitrealinjection.

Systemic administration can also be transmucosal or transdermal. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art. The compounds can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In addition to the other forms of delivery, the compounds aredeliverable via eye drop or intraocular injection. With respect to eyedrops, the compositions of the present disclosure optionally compriseone or more excipients intended for topical application to the eye ornose. Excipients commonly used in pharmaceutical compositions intendedfor topical application to the eyes, such as solutions or sprays,include, but are not limited to, tonicity agents, preservatives,chelating agents, buffering agents, surfactants and antioxidants.Suitable tonicity-adjusting agents include mannitol, sodium chloride,glycerin, sorbitol and the like. Suitable preservatives includep-hydroxybenzoic acid ester, benzalkonium chloride, benzododeciniumbromide, polyquaternium-1 and the like. Suitable chelating agentsinclude sodium edetate and the like. Suitable buffering agents includephosphates, borates, citrates, acetates and the like. Suitablesurfactants include ionic and nonionic surfactants, though nonionicsurfactants are preferred, such as polysorbates, polyethoxylated castoroil derivatives and oxyethylated tertiary octylphenol formaldehydepolymer (tyloxapol). Suitable antioxidants include sulfites, ascorbates,BHA and BHT. The compositions of the present disclosure optionallycomprise an additional active agent. With the exception of the optionalpreservative ingredient (e.g., polyquaternium-1), the compositions ofthe present disclosure preferably do not contain any polymericingredient other than polyvinylpyrrolidone or polystyrene sulfonic acid.

When the compositions of the present disclosure containpolyvinylpyrrolidone, the polyvinylpyrrolidone ingredient is preferablyselected or processed to minimize peroxide content. Freshly producedbatches of polyvinylpyrrolidone are preferred over aged batches.Additionally, particularly in cases where the composition will containgreater than 0.5% polyvinylpyrrolidone, the polyvinylpyrrolidoneingredient should be thermally treated (i.e., heated to a temperatureabove room temperature) prior to mixing with olopatadine in order toreduce the amount of peroxides in the polyvinylpyrrolidone ingredientand minimize the effect of peroxides on the chemical stability ofolopatadine. While thermally treating an aqueous solution ofpolyvinylpyrrolidone for prolonged periods will substantially reduce theamount of peroxides, it can lead to discoloration (yellow toyellowish-brown) of the polyvinylpyrrolidone solution. In order tosubstantially reduce or eliminate peroxides without discoloring thepolyvinylpyrrolidone solution, the pH of the aqueous solution ofpolyvinylpyrrolidone should be adjusted to pH 11-13 before it issubjected to heat. Much shorter heating times are needed to achievesignificant reductions in peroxide levels if the pH of thepolyvinylpyrrolidone solution is elevated.

One suitable method of thermally treating the polyvinylpyrrolidoneingredient is as follows. First, dissolve the polyvinylpyrrolidoneingredient in purified water to make a 4-6% solution, then raise the pHof the solution to pH 11-13, (an effective range of pH is 11-11.5), thenheat to a temperature in the range of 60-121° C., preferably 65-80° C.and most preferably 70-75° C. The elevated temperature should bemaintained for approximately 30-120 minutes (preferably 30 minutes).After the heated solution cools to room temperature, add HCl to adjustthe pH to 3.5-8, depending upon the target pH for the olopatadinecomposition.

Particularly for compositions intended to be administered as eye drops,the compositions preferably contain a tonicity-adjusting agent in anamount sufficient to cause the final composition to have anophthalmically acceptable osmolality (generally 150-450 mOsm, preferably250-350 mOsm). The ophthalmic compositions of the present disclosurepreferably have a pH of 4-8, preferably a pH of 6.5-7.5, and mostpreferably a pH of 6.8-7.2.

The eye-drop compositions of the present disclosure are preferablypackaged in opaque plastic containers. A preferred container for anophthalmic product is a low-density polyethylene container that has beensterilized using ethylene oxide instead of gamma-irradiation.

With respect to opthamalic injectables, the pharmaceutical compositionsof this disclosure are administered to the area in need of treatment bysubconjunctival administration. One preferred method of subconjunctivaladministration to the eye is by injectable formulations comprising thepharamaceutical compositions disclosed herein. Another preferred methodof subconjunctival administration is by implantations comprising slowreleasing compositions.

Compositions that are delivered subconjunctivally comprise, according toembodiments, an ophthalmic depot formulation comprising an active agentfor subconjunctival administration. According to embodiments, theophthalmic depot formulation comprises microparticles of essentiallypure active agent. The microparticles comprising can be embedded in abiocompatible pharmaceutically acceptable polymer or a lipidencapsulating agent. The depot formulations may be adapted to releaseall of substantially all the active material over an extended period oftime. The polymer or lipid matrix, if present, may be adapted to degradesufficiently to be transported from the site of administration afterrelease of all or substantially all the active agent. The depotformulation can be liquid formulation, comprising a pharmaceuticalacceptable polymer and a dissolved or dispersed active agent. Uponinjection, the polymer forms a depot at the injections site, e.g., bygelifying or precipitating.

Solid articles suitable for implantation in the eye can also be designedin such a fashion to comprise polymers and can be bioerodible ornon-bioerodible. Bioerodible polymers that can be used in preparation ofocular implants carrying the compositions of the present disclosureinclude without restriction aliphatic polyesters such as polymers andcopolymers of poly(glycolide), poly(lactide), poly(ε-caprolactone),poly(hydroxybutyrate) and poly(hydroxyvalerate), polyamino acids,polyorthoesters, polyanhydrides, aliphatic polycarbonates and polyetherlactones. Illustrative of suitable non-bioerodible polymers are siliconeelastomers.

According to embodiments, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the disclosure, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

The term “effective amount” refers to an amount of a biologically activemolecule or conjugate or derivative thereof sufficient to exhibit adetectable therapeutic effect without undue adverse side effects (suchas toxicity, irritation and allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of the invention.The therapeutic effect may include, for example but not by way oflimitation, being substantially cytotoxic to cancer cells, but lesscytotoxic to natural cells. The effective amount for a subject willdepend upon the type of subject, the subject's size and health, thenature and severity of the condition to be treated, the method ofadministration, the duration of treatment, the nature of concurrenttherapy (if any), the specific formulations employed, and the like.Thus, it is not possible to specify an exact effective amount inadvance. However, the effective amount for a given situation can bedetermined by one of ordinary skill in the art using routineexperimentation based on the information provided herein.

EXAMPLES

A more complete understanding of the present disclosure can be obtainedby reference to the following specific examples and figures. Theexamples and figures are described solely for purposes of illustrationand are not intended to limit the scope of the disclosure. Changes inform and substitution of equivalents are contemplated as circumstancesmay suggest or render expedient. Although specific terms have beenemployed herein, such terms are intended in a descriptive sense and notfor purposes of limitations. Modifications and variations of thedisclosure as hereinbefore set forth can be made without departing fromthe spirit and scope thereof, and, therefore, only such limitationsshould be imposed as are indicated by the appended claims.

Example 1 F-HSA Exhibits Enhanced Fluorescence Levels ofAmyloid-Specific Dye ThT in a Dose-Dependent Manner

FIG. 1 is an implementation of experimental data shows that F-HSA, likeamyloid fibrils A β (1-42), exhibit enhanced fluorescence level ofamyloid-specific dye ThT in a dose-dependent manner as compared with BSAnot processed by the Superdex-200 column. This result shows that F-HSAhas a fibrillar structure like A β (1-42), whereas HSA has a globularstructure. (Binding to ThT is one of the characteristics of amyloid-likeproteins.)

Example 2 F-HSA has a Cytotoxic Effect on Prostate Cancer Cells

FIG. 2 shows F-HSA's cytotoxic effect in TS/A cells and FIG. 3 showsF-HSA's cytotoxic effect in MDA-MB-231 cells. Each respective cell typewas treated for 16 h in serum-free culture medium with variousconcentrations of F-HSA. Cell viability was determined by the MTT assay.Globular HSA has no cytotoxic effect on normal or cancer cells.

Example 3 F-HSA has a Cytotoxic Effect on Prostate Cancer Cells

FIG. 4 shows F-HSA's cytotoxic effect in SKOV-3 cells and FIG. 5 showsF-HSA's cytotoxic effect in CaSki cells. Each respective cell type wastreated for 16 h in serum-free culture medium with variousconcentrations of F-HSA. Cell viability was determined by the MTT assay.

Example 4 F-HSA has a Cytotoxic Effect on Prostate Cancer Cells

FIG. 6 shows F-HSA's cytotoxic effect in PC-3 cells and FIG. 7 showsF-HSA's cytotoxic effect in 22 Rvl cells. The respective cell type wastreated for 16 h in serum-free culture medium with variousconcentrations of F-HSA. Cell viability was determined by the MTT assay.

Example 5 F-HSA has a Cytotoxic Effect on Lung Cancer Cell Lines

According to implementations shown in FIG. 8, F-HSA was shown to inducecytotoxicity in adenocarcinoma cell lines A549, CL1-0, Cl1-5, H1299,PC13, and PC14; squamous cell carcinoma lung cancer cell line H520, andlarge cell lung cancer carcinoma cell line H661. FIG. 8 shows the IC₅₀of each of the respective cell lines.

Example 6 F-HSA Suppresses Tumor Cell Invasion and Migration In Vitro

F-HSA was also shown to be effective in suppressing tumor cell invasionand migration in vitro, as shown according to implementations ofexperimental data in FIG. 9. As shown in FIGS. 9A, 9C, 9E, and 9G, F-HSAsignificantly reduced the tumor cells invasion/migration abilities, atconcentrations which did not affect viability of either cancer or normalcells.

Example 7 F-HSA Suppressed Tumor Cell Metastasis In Vivo

F-HSA suppressed breast cancer tumor cell lines TS/A and MDA-MB-231 invivo. Breast cancer cells were injected via the tail vein of thesubjects. Tumor cell foci detected in the lung tissue indicated that thebreast cancer cells had metastasized into lung. FIGS. 10A and 11A showF-HSA significantly suppressed the metastasis of breast cancer TS/Acells and MDA-MB-231 cells to lung compared with TS/A or MDA-MB-231bearing mice without F-HSA treatment. FIGS. 10B, 10C, 11B, and 11Cmeasure the weight and the number of tumor cell foci in the lungtissues, which further confirmed the efficacy of F-HSA in vivo.

Example 8 Materials and Methods

Preparation of F-HSA.

Twenty milligrams of HSA was dissolved in 10 ml of PBS with 1% SDS(w/v). The HSA solution was sonicated for 5 min and subsequently appliedto a Superdex-200, which was previously equilibrated with the elutingsolution (25 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 M NaCl, and 0.05%SDS). The column was eluted at the rate of 1 ml/min and fractions C3 toC7 that contained HSA were pooled. The pooled fractions wereconcentrated to 2˜3 mg/ml then dialyzed against PBS with Cellu-SepT4/Nominal (MWCO: 12,000-14,000 Da) dialysis membrane. New PBS bufferwas exchanged every two hours at room temperature three times. The yieldof the HSA-S200 was about 75%.

Thioflavin T (ThT) Fluorescence Assay.

Binding to ThT is one of the characteristics of amyloid-like proteins.For fluorescence measurements, increasing concentrations of proteinswere incubated with 20 μM ThT for 1 h at room temperature, thefluorescence was then measured in triplicate on a Wallac Victor² 1420Multilabel Counter (Perkin Elmer Life Science, Waltham, Mass., USA).Excitation and emission wavelengths were 430 nm and 486 nm,respectively. ThT background signal from buffer solution was subtractedfrom corresponding measurements.

Cell Survival was Determined by MTT Colorimetric Assay.

Exponentially growing cells (2×10⁴ cells/well for TS/A) were seeded in a96-well plate in medium with 10% FBS and incubated for 24 h. Treatmentof cells with a series of concentrations of proteins was carried out inserum-free medium for 16 hr indication at 37° C. After treatment, MTTsolution was added to each well (0.5 mg/ml), followed by a 4 hincubation period. The viable cell number is directly proportional tothe production of formazan, which, following solubilization withisopropanol, can be measured spectrophotometrically at 570 nm by anELISA plate reader.

Cell Viability Assays.

Cell viability was measured by WST-1 assay according to themanufacturer's instructions (Roche, Mannheim, Germany). In brief, 2×10⁴cells were added to 100 μl media per well on a 96 well plate andincubated at 37° C. in 5% CO₂ overnight in a humidified incubator. Thecells attached to the wells were incubated in serum-free medium andtreated with various concentrations of F-HSA. After incubation at 37° C.in 5% CO₂ for 16 h to allow the drug to take effect, 10 μl WST-1 reagentwas added to each well. The plate was then placed onto a shaking tableand shaken at 150 rpm for 1 min. After incubation at 37° C. in 5% CO₂for another 2 h to allow the WST-1 reagent to be metabolized, theproportion of surviving cells were determined by optical density (450 nmtest wavelength, 690 nm reference wavelength). The percentage ofsurviving cells was calculated as (O.D. treatment/O.D. control)×100%while the percentage of growth inhibition was calculated as [1−(O.D.treatment/O.D. control)]×100%. According to this experiment, IC₅₀ is theconcentration at which the reagent yields 50% inhibition of the cellularviability.

Cell Migration and Invasion Assays.

Cell migration and invasion were determined by using Boyden chambermigration and invasion assay (Corning). In brief, the 8-μm poremembranes of the upper chambers were coated with 20 μg/ml fibronectin(for cell migration assay) or 40 μg/ml Matrigel (for cell invasionassay) and placed in a well with 1 ml of PBS and incubated for 2 h at37° C. Cancer cells (1×10⁵) in 100 μl of serum free culture medium wereseeded in the upper chamber for 1 h. A serially diluted concentration ofF-HSA was added into the upper chamber and then the culture mediumcontaining 10% FBS was added to the lower chamber. Cells were incubatedfor 24 h at 37° C. After incubation, cells on the upper side of themembrane were removed by wiping it with a cotton swab, and cells thathad migrated onto the lower membrane surface were dissociated by usingcell dissociation solution (Sigma) and counted by flow cytometer (BDcompany). At these concentrations, however, F-HSA did not affect cellviability when measured with the MTT assay, and as shown in FIGS. 9B,9D, 9F, and 9H. Viability and cytotoxicity was measured using MTT orWST-1 assay. These kits are designed for the spectrophotometricmeasurement of cell growth as a function of mitochondrial activity inliving cells (Roche).

Breast Cancer Cell Metastasation In Vivo.

TS/A murine mammary adenocarcinoma cells were intravenously injectedinto the lateral tail vein of BALB/c mice or MDA-MB-231 human mammaryadenocarcinoma cells were injected into nude mice. F-HSA (1 mg/kg) wasthen injected intravenously at next day and then one time every two daysfor ten times. At the end of F-HSA treatment, mice were sacrificed todetect the metastasis of lungs.

While the method and agent have been described in terms of what arepresently considered to be the most practical and preferredimplementations, it is to be understood that the disclosure need not belimited to the disclosed exemplary implementations it is apparent thatmodifications and adaptations of those implementations will occur tothose skilled in the art. It is intended to cover various modificationsand similar arrangements included within the spirit and scope of theclaims, the scope of which should be accorded the broadestinterpretation so as to encompass all such modifications and similarstructures. The present disclosure includes any and all implementationsof the following claims.

The invention claimed is:
 1. A composition comprising: fibrillar humanserum albumin and a pharmaceutically acceptable carrier wherein thehuman serum albumin is produced by a method comprising: dissolving humanserum albumin in a suitable detergent sonicating the human serum albuminapplying the sonicated human serum albumin to a gel filtration columnwith a separation range above 70 kDA molecular weight; so as to promotecolumn-induced fibrillar protein formation and to separate globularprotein from fibrillar protein; eluting the fibrillar human serumalbumin from the column with a buffer containing a low concentration ofdetergent; collecting eluent containing fibrillar human serum albuminfrom the column, and dialyzing the eluent to remove the detergent.